Human stem cells have traditionally been grown over layers of feeder cells because fibroblast feeder cells secrete as yet unknown factors that increase growth and inhibit spontaneous differentiation of stem cells. Later, in an effort to develop defined surfaces that enable stem cell growth, Matrigel was identified as a surface coating that supported stem cell growth if used in conjunction with bFGF and conditioned media (cell secretions) from fibroblast feeder cells. In an improvement, the present inventor previously determined that conditioned media from feeder cells was not required for stem cell growth, on Matrigel, if the stem cell growth media contained a MUC1* activator such as bivalent anti-MUC1* antibody or NM23 in dimeric form, preferably a mutant, such as NM23-S120G that preferentially forms dimers, while resisting the characteristic formation of tetramers and hexamers.
However, although stem cell growth over a layer of Matrigel is an improvement over a cell-based surface, it is not a defined or xeno-free surface, which is the end goal for the growth of human stem cells destined for therapeutic use. Matrigel is a mixture of components that are not desirable for cells destined for human transplant. Matrigel contains among other things mouse sarcoma cells. Therefore, those in the field appreciate that what is needed is a surface for stem cell growth that is defined and preferably xeno-free (free of animal material).
Several surfaces that are defined and xeno-free have been reported and some are commercially available. Vita™ surface (ThermoFisher, USA), hydrogel coated surfaces, and recombinant Vitronectin have been reported to facilitate stem cell attachment and growth. However they still require the use of feeder cell conditioned media. In addition, the degree of stem cell attachment has in general been less than what Matrigel supports. Another problem that plagues this field is that whenever stem cell growth media or surfaces are changed, the stem cells must adapt gradually. This period of adaptation can take weeks to months to change stem cell media or growth surface.
Recent research indicates that the mechanical nature of a surface impacts a stem cell's ability to remain pluripotent. For example, rigid surfaces have been shown to induce differentiation whereas more flexible surfaces inhibit spontaneous differentiation. Pressure is another factor that affects stem cell differentiation or resistance to differentiation. In addition to the mechanical characteristics of surfaces, the chemical nature of a surface has been shown to affect differentiation. Further, it has been reported that stem cells of different stages of differentiation have different binding preferences. That is, stem cells at one stage may attach and grow on a surface having certain chemical characteristics while stem cells at another stage do not bind to the first surface but attach and grow on a second surface having different chemical makeup than the first surface.
Therefore it would be an improvement over existing methods to develop defined surfaces for human stem cell growth and maintenance that enable stem cell attachment, and also promote pluripotent stem cell growth. A further improvement to the state of the art would be if these defined growth surfaces bound to ligands known to promote pluripotency. An even further improvement would be if a surface and growth media were developed to make an entirely defined system for pluripotent stem cell growth, even more preferred if the system could be free of animal products. It would be a vast improvement over the state of the art if methods could be identified that streamline stem cell adaptation so that growth media or growth surfaces can be changed without the typical 4-8 week acclimation period.
Recently researchers (J. Nichols and A. Smith, Cell Stem Cell 4 (6), 487 (2009), J. Hanna, A. W. Cheng, K. Saha et al., Proc Natl Acad Sci USA 107 (20), 9222 (2010).) reported that human stem cells grown by conventional methods are not truly pluripotent stem cells, but have already undergone differentiation to a more mature state called “primed.” Primed stem cells grow via the bFGF/TGF-beta pathway and closely resemble mouse stem cells derived from the epiblast rather than the “naïve” or “ground state” mouse stem cells that are derived from the inner cell mass. The consensus from the early research in the area of naïve versus primed human stem cells is that: 1) human naïve stem cells are not stable in the presence of bFGF; and 2) the growth factors or pathway by which human naïve stem cells grow is as yet unknown.
Research has now shown that human stem cells cultured in bFGF containing media are no longer truly pluripotent (J. Hanna, A. W. Cheng, K. Saha et al., Proc Natl Acad Sci USA 107 (20), 9222 (2010)). In a watershed research article, Jaenisch and colleagues describe human embryonic stem (ES) cells as being “primed” rather than being true pluripotent stem cells, which they term “Naïve”. Research has now shown that human stem cells in the naïve state cannot be maintained in standard stem cell growth media wherein the major growth factor is bFGF.
By comparing human ES cells to mouse ES cells wherein both were derived from the blastocyst-stage embryos, the researchers discovered that the human ES cells were morphologically and molecularly different from the mouse stem cells. They further disclosed that the human ES cells that have been isolated thus far are not truly pluripotent and more closely resemble mouse stem cells that have been derived from the epiblast stage which is a later stage of development. These findings and others indicate that what we think of as human pluripotent ES cells are actually more mature than true pluripotent stem cells. Jaenisch and colleagues discovered molecular markers that identify naïve stem cells and markers that identify primed stem cells.
Researchers were able to temporarily make human primed stem cells revert to the naïve state by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Forskolin, a protein kinase A pathway agonist which can induce
Klf4 and Klf2 expression, transiently replaced the need for ectopic expression of those two genes. Once the human ES cells had been reverted to the naïve state, they needed to be cultured in PD/CH/LIF/FK but could only remain naïve for a few passages before they matured to primed cells. This is strong evidence that the researchers were not able to identify the growth factors that promote and maintain human ES cells in the pluripotent naïve state. In contrast to conventional human ESCs, these epigenetically converted naïve stem cells gained expression of Oct4, Nanog, Klf4, Klf2, Tbx3, Gbx2, Lin28 and SOCS3 (Naïve markers), and lost or had greatly reduced expression of Otx2, Sox17, Cer1, Foxa2, Zic1, Lhx2 and XIST (Primed markers). In addition, primed cells that were transiently reverted to the naïve state grew in sheets rather than in colonies.
However, Nichols and Smith report that the Naïve markers are Oct4, Nanog, Klf4, Klf2, Rex 1 and NrOb 1 and that naïve cells had lost or had greatly reduced expression of FGF5 and markers of X-inactivation such as XIST. The discrepancy between the lists of naïve markers and primed markers generated by these two research teams may be due differences in the naïve stem cells they were analyzing; Hanna et al analyzed primed human stem cells that they had transiently reverted to the naïve state, determined by their similarity to mouse naïve stem cells. Alternatively, genes identified by the earlier research may cause activation of the genes identified in the later, more extensive studies described in Hanna et al. Both studies agree that the naïve markers consist at least of Oct4, Nanog, Klf4 and Klf2, and the primed markers consist at least of FOXa2 and XIST.
Previous research has not been able to identify the growth pathway or the growth factor(s) that made human stem cells propagate as naïve stem cells. Further, even with ectopic expression of genes and growth in a concoction of factors, the reverted-naïve cells remained naïve for a short period of time and then progressed to the more differentiated primed stage.
It would be a significant improvement if one could identify methods for cultivating naïve stem cells. Such methods would include identification of the growth pathways that stimulate growth and maintenance of the naïve state, development of media that enables their proliferation, or identification of surfaces that naïve stem cells bind to for growth or isolation of naïve stem cells.
Therefore what is needed is a method for propagating human stem cells as naïve stem cells directly after harvest from either an embryo or from an induced pluripotent state, or a method to revert primed stem cells to the naïve state and then maintain them in that state for prolonged periods of time. What is needed is a method for stably converting primed stem cells to the naïve state, whereas current methods can only transiently hold the cells in the naïve state. Ideally, the method for maintaining human stem cells in the naïve state or converting them from the primed state to the naïve state would not involve ectopic expression of genes.